This invention relates to the removal of endotoxin from biological fluids including the blood of animals and parenteral fluids, such as serum, plasma, whole blood, albumins, dextrose solutions, and the like. More particularly, the invention pertains to a novel process for removing endotoxin from such biological fluids through the use of certain nonionogenic hydrophobic synthetic plastic polymers or resins that are capable of adsorbing endotoxin present in the fluids.
Generally speaking, endotoxin is a complex lipopolysaccharide material derived from gram-negative bacilli that is known to produce a wide variety of striking pathophysiological reactions in aminals. Studies have demonstrated that endotoxin is distinguishable from classic protein toxins due to its failure to be neutralized by anti-serum, is increased heat stability and its failure to be converted to toxoid by treatment with formaldehyde. Moreover, endotoxin exhibits a lesser degree of potency than classic protein toxins and produces essentially similar reactions in animals regardless of the microbial orgin of the endotoxin. The material has been known and studied for many years particularly in regard to the pathophysiological reactions it causes in animals. For many years it was believed that the material was contained within gram-negative bacilli cells and was released only upon disintegration of the cell walls. Hence, the material was termed endotoxin. Recent studies, however, have shown that endotoxin is localized at the cell surface of gram-negative bacilli and may be present with viable and killed cells as well as in a free form within a liquid medium.
So far as is presently known, the complex lipopolysaccharide material commonly identified as endotoxin may be derived from all types of gram-negative bacilli including, by way of example, the Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Citrobacter, Bordetella, Serratia and Shigella types, to name a few. Endotoxins derived from different types of gram-negative bacilli are essentially the same in biochemical composition and structure and, as mentioned above, produce essentially similar reactions in animals.
As mentioned before, endotoxin is known to cause several striking and varied pathophysiological reactions and has been identified as a direct and contributory cause of death of many hospitalized patients. More particularly, endotoxin is known to cause febrile reactions in animals with symptons of extremely high fever, vasodilation, diarrhea, and the like and, in extreme cases, fatal shock. It is also known that endotoxin cause leucocytosis, deleterious changes in carbohydrate and protein metabolism and widespred intravascular clotting by fibrin formation.
Studies have shown that endotoxemia in animals may be caused by or is associated with gram-negative bacilli primary and secondary infections and/or the employment of intravenous apparatus or solutions contaminated with gram-negative bacilli or endotoxin. The occurrence of endotoxemia from the use of endotoxin-contaminated intravenous or parenteral solutions has recently been recognized as a particular problem in modern hospitals. In addition, it has recently been found that severe trauma, particularly trauma caused by thermal injuries, may cause the release of endotoxin from gram-negative bacilli of the noram flora of the gastrointestinal tract of animals. The studies have shown that there may be increased levels of endotoxin in the blood of traumatized animals even when the animals have no other diagnosed bacterial infection.
Under normal conditions the blood cells, i.e., leucocytes, of animals usually control the level of endotoxin in the blood. However, the blood cells usually cannot control excessive amounts of endotoxin of endotoxin experienced under abnormal conditions, such as those hereinabove mentioned, thereby resulting in endotoxemina. It is presently a common practice in the medical profession to counteract endotoxemia by treatment with massive infusions of antibiotics. However, it has not been shown that antibiotics remove endotoxin other than by controlling gram-negative bacilli. As mentioned hereinbefore, endotoxin is known to exist in free form in liquid media and may be associated with killed bacterial cells.
There are a few procedures known for removing or reducing the level of endotoxin in certain fluid media. For example, endotoxin may be removed from a liquid medium by filtration procedures employing macromolecular and/or activated carbon filters whereby the complex endotoxin molecules are filtered out. Osmotic pressure separation procedures have also been employed. These techniques have generally been employed in the purification of water and relativelysimple fluid compositions. However, such techniques have not been extensively used to remove endotoxin from biological fluids, particularly certain parenteral fluids, such as plasma, serum, albumins, whole blood and the like, apparently due to the extremely complex molecular and sometimes cellular composition of such fluids. In fact, it is common practice in the medical and pharmacological professions to merely destroy parenteral fluids contaminated with unacceptable levels of endotoxin.
I have now discovered a process for selectively removing endotoxin from substantially any biological fluid which does not otherwise adversely affect the molecular and/or cellular composition of the fluid. In fact, the process of my invention is particularly useful in removing and/or reducing the level of endotoxin in the blood of animals in accordance with in vivo hemoperfusion techniques. The inventive process is based upon the surprising discovery that certain non-ionogenic hydrophobic synthetic plastic polymers have specific affinity for endotoxin when placed in intimate contact therewith.
Several types of synthetic plastic resins or polymers have heretofore been used in various processes for treating parenteral fluids and/or blood. For example, there are several known procedures for treating parenteral fluids by the employment of ion-exchange resins. More particularly, ion-exchange resins have been employed in processes for treating parenteral fluids, including blood, with anionic and cationic agents, for separating certain protenatious materials from blood, for preparing sterile parenteral fluids difficult to sterilize, such as bicarbonate ion solutions, and the like. See U.S. Pat. Nos. 3,769,401; 3,097,141; 3,234,199; 2,682,268; and 3,305,446 to name a few. The ion-exchange resins employed in these processes are basically comprised of monomers and/or polymers of styrene or vinyl benzene treated with many types of polyelectrolytes.
It is known that certain types of strong basic anion exchange resins have an affinity for bacterial endotoxin. James B. Nolan and M. Vilawat Ali, in "Effect Of Cholestyramine On Ednotoxin Toxicity And Absorption", American Journal Of Digestive Diseases, Vol. 17, No. 2 (February, 1972), have reported that the addition of cholestyramine or DOWEX 1-X8 (Dow Chemical Company), both strong basic anion exchange resins, to an endotoxin mixture impedes its absorption in the isolated gut sac, and reduces its toxicity when injected intraperitonially. However, it has not been reported that the described ion-exchange resins are capable of selectively absorbing and removing bacterial endotoxin from complex biological fluids, such as albumin, blood, etc. As known, such ion-exchange resins are strongly ionogenic and, thus, may adversely affect such complex biological fluids. Furthermore, such resins are completely different types of resins, chemically, physically, etc., from the certain types of non-ionogenic hydrophobic synthetic plastic polymers described below which I have found to have specific affinity for bacterial endotoxin when placed in intimate contact therewith.
U.S. Pat. No. 3,794,584 teaches a process for removing poisonous or toxic amounts of barbiturates and glutethimides from blood which includes perfusing blood over a column of an essentially non-ionogenic macroreticular watersoluble cross-linked polymer having a porosity of at least 10% and a specific surface area of at least 10 square meters per gram. The polymer resin employed is described as being comprised of from 2 to 100 weight percent of a poly(vinyl)benzene monomer polymerized with one or more mono- or polyethylenically unsaturated monomers. The disclosed poly(vinyl)benzene-based macroreticular polymer resins are described as being capable of adsorbing the barbiturates and glutethimides from the blood without otherwise adversely affecting the blood.
U.S. Pat. No. 3,706,661 teaches a method for the separation of biological cells from solutes by use of macroporous synthetic plastic resin gels, particularly gels of polyacrylamide and hydrophilic polymethacrylates. U.S. Pat. No. 3,839,314 describes a process of clarifying blood serum and plasma to remove undesired protenatious and lipid matter by the employment of block copolymers of ethylene oxide and a polyoxypropylene polymer. There are also several prior art references which describe the use of certain synthetic plastic resins, such as nylon, acrylonitrile polymers, polyesters, and polytetrafluoroethylene in fiber or textile form as filter media to remove materials from certain parenteral fluids. See U.S. Pat. Nos. 3,462,361; 3,448,041; 3,036,575; 3,533,400; and 2,702,036.
However, to my knowledge, the synthetic polymeric resins that I have found to be capable of adsorbing endotoxin have not heretofore been specifically employed in any prior art processes for treating biological fluids, particularly for removing endotoxin. Furthermore, many types of synthetic resins heretofore employed in processes for treating biological fluids to remove certain components have been found to have no affinity for endotoxin.